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Thermal proteome profiling of stationary-phase E. coli treated with semapimod

ABSTRACT: There is a need to discover and develop non-toxic antibiotics that are effective against metabolically dormant bacteria, which underlie chronic infections and promote antibiotic resistance. Traditional antibiotic discovery has historically favored compounds effective against actively metabolizing cells, a property that is not predictive of efficacy in metabolically inactive contexts. Here, we combine a stationary-phase screening method with deep learning-powered virtual screens and toxicity filtering to discover compounds with lethality against metabolically dormant bacteria and favorable toxicity profiles. The most potent and structurally novel compound without any obvious mechanistic liability was semapimod, an anti-inflammatory drug effective against stationary-phase E. coli and A. baumannii. Integrating microbiological assays, biochemical measurements, and single-cell microscopy, we show that semapimod selectively disrupts and permeabilizes the bacterial outer membrane by binding lipopolysaccharide. This work illustrates the value of harnessing non-traditional screening methods and deep learning models to identify non-toxic antibacterial compounds that are effective in infection-relevant contexts.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Escherichia Coli

SUBMITTER: Alice Herneisen

LAB HEAD: Sebastian Lourido

PROVIDER: PXD044230 | Pride | 2023-08-21

REPOSITORIES: Pride

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			Action	DRS
	20230218_Exploris_ES902_LouridoLab_AK_DMSO_Fraction_.mzML	Mzml
	20230218_Exploris_ES902_LouridoLab_AK_DMSO_Fraction_.mzid.gz	Mzid
	20230218_Exploris_ES902_LouridoLab_AK_DMSO_Fraction_1.raw	Raw
	20230218_Exploris_ES902_LouridoLab_AK_DMSO_Fraction_2.raw	Raw
	20230218_Exploris_ES902_LouridoLab_AK_DMSO_Fraction_3.raw	Raw

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Project description:The DosR regulon in Mycobacterium tuberculosis is involved in respiration-limiting conditions and its induction is controlled by two histidine kinases, DosS and DosT. Recent experimental evidence indicates DosS senses either molecular oxygen or a redox change. This report demonstrates that DosS responds to a reduced electron transport system (ETS), although this does not rule out a role for oxygen in silencing signaling. Under aerobic conditions induction of the DosR regulon by DosS but not DosT was observed after the addition of ascorbate, a powerful cytochrome c reductant, demonstrating DosS responds to a redox signal even in the presence of high oxygen tension. During hypoxic conditions regulon induction was attenuated by treatment with compounds that occluded electron flow into the menaquinone pool or decreased the size of the menaquinone pool itself. Increased regulon expression during hypoxia was observed when exogenous menaquinone was added, demonstrating the menaquinone pool is a limiting factor in regulon induction. Taken together these data indicate that a reduced menaquinone pool directly or indirectly triggers induction of the DosR regulon via DosS. Biochemical analysis of menaquinones upon entry into hypoxic/anaerobic conditions demonstrated the disappearance of the unsaturated species and low-level maintenance of the mono-saturated menaquinone. Relative to the unsaturated form, an analog of the saturated form is better able to induce signaling via DosS, rescue inhibition of menaquinone synthesis, and is less toxic. The menaquinone pool is central to the ETS and therefore provides a mechanistic link between the respiratory state of the bacilli and DosS signaling. Aerobically growing logarithmic phase DosS and DosT mutant strains were analyzed after treatment with the cytochrome c reductant ascorbate to examine the effect on DosR signaling caused by reducing the electron transport system. Experiments were repeated in triplicate (dosT mutant) or duplicate (dosS mutant).

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Thermal proteome profiling of stationary-phase E. coli treated with semapimod

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